Structural insights into substrate recognition in proton-dependent oligopeptide transporters

Abstract

Short-chain peptides are transported across membranes through promiscuous proton-dependent oligopeptide transporters (POTs)--a subfamily of the major facilitator superfamily (MFS). The human POTs, PEPT1 and PEPT2, are also involved in the absorption of various drugs in the gut as well as transport to target cells. Here, we present a structure of an oligomeric POT transporter from Shewanella oneidensis (PepTSo2), which was crystallized in the inward open conformation in complex with the peptidomimetic alafosfalin. All ligand-binding residues are highly conserved and the structural insights presented here are therefore likely to also apply to human POTs.

Figure 1

Structure of the proton-dependent oligopeptide transporter PepT_So2. The transporter was crystallized in an inward open conformation in complex with the compound alafosfalin. (A) The overall structure of PepT_So2 viewed from the plane of the membrane. N- and C-terminal subdomains are coloured yellow and blue respectively, helices HA and HB are coloured grey. Approximate dimensions of the molecule are presented and black bars depict the approximate location of the membrane. Alafosfalin, shown as red spheres, is buried in the central binding pocket located between the N- and C-terminal subdomains. (B) Schematic model of the MFS POT alternating access transport mechanism. The N- and C-bundles are coloured blue and red respectively. The substrate is presented as a black square located in the binding pocket. Available POT structures depicting different stages of the transport cycle are placed next to the schematic figures; helices HA and HB are coloured grey. (C) Overview of available POT structures including nomenclature, PDB codes and source organism. MFS, major facilitator superfamily; POT, proton-dependent oligopeptide transporter.

Figure 2

PepT_So2-binding pocket and alafosfalin coordination. (A) The omit density, Fo-Fc, of alafosfalin is shown in green and contoured at 3 σ. Highly conserved amino-acid residues in the binding pocket are labelled and shown as sticks. (B) 2Fo-Fc density for alafosfalin and the conserved residues in the binding pocket; electron density map is shown in blue and contoured at 1 σ. The flexible nature of K121 resulted in poor electron density for the side chain, this is illustrated as a dashed circle. (C) Electrostatic surface of PepT_So2 showing the location and dipole like charge distribution of the binding site. The alafosfalin phosphate head group is pointing towards the positively charged surface of the binding pocket whereas the N-terminus is oriented closer towards the negative surface. (D) Chemical formula of alafosfalin. (E) Thermal shift assay monitoring the degree of precipitation using centrifugation after unfolding (see supplementary information online). Coomassie stained gels of PepT_So2 incubated with ±10 mM alafosfalin and heated up to 70 °C. (F) Resulting melting curves show a stabilization of PepT_So2 incubated with alafosfalin compared to the control samples, indicative of ligand binding. (G) Stabilization of PepT_So2 in the presence of different peptides and compounds. The stabilization effect is normalized against the stability of apo PepT_So2. The sugar transporter XylE, which has a similar thermal stability as PepT_So2, was used as a control protein and the results are shown. Error bars represent standard deviation from triplicate experiments.

Figure 3

Gating residues and structural comparison of PepT_So2, PepT_So and PepT_St. (A) Two conserved networks of hydrogen bonds and salt bridges (coloured blue and red), which might act as potential gates are found on the periplasmic side of PepT_So2. Y37 (yellow) mediates more interactions between the N- and C-terminal bundles, blocking access to the binding site from the periplasm. Corresponding residues are labelled and shown as sticks. (B) Conformational changes of H11 between occluded and inward open structures. The position of the respective methionine residues, regulating access to the binding site from the cytoplasm, is presented as sticks. H11 of PepT_So2 is shown in blue, PepT_So in green and PepT_St in brown. (C, D) Cartoon representation of PepT_So2 in blue, PepT_So in green and PepT_St in brown. N- and C-terminal bundles and individual helices are labelled. The structures were superimposed on the N-terminal subdomain and shown from the periplasmic side (C) and cytoplasmic side (D). Arrows denote movements of individual helices in the C-subdomain relative to the PepT_So occluded-state structure. The additional linker helices HA and HB are omitted for clarity.

Figure 4

Oligomeric structure of PepT_So2. (A) Analytical size-exclusion chromatograms of four POT transporters suggest that PepT_So2 has the largest molecular weight. Elution profiles for different POTs are color coded according to the legend displayed on the right part of the panel. (B) SDS–PAGE of glutaraldehyde cross-linked PepT_So2, PepT_So and PepT_St purified in the detergent DDM. The different oligomeric states are indicated by arrows and asterisks. The incubation time ranged from 0–15 min. (C) BN-PAGE of five different prokaryotic POT transporters indicates different oligomeric arrangements. (D) Negative stain EM of PepT_So2 tetramers. In the upper panel, the tetrameric nature of the particles is visible in an otherwise clearly monodisperse preparation. The scale bar represents 200 nm. The lower panel shows a gallery of 11 classes viewing the tetramer slightly tilted compared to or parallel to the membrane plane with dimensions of about 12.4 × 12.4 nm. The frame size of the boxed, magnified particles is 21.4 nm. (E) Surface representation of a potential tetramer arrangement obtained from a different crystal form (P3₁21) at lower resolution. The helices HA and HB are coloured grey to facilitate the visualization of four-fold symmetry. (F) The P3₁21 tetramer is shown as cylinders in magenta, H12 is coloured orange. A PepT_So2 monomer derived from the P2₁2₁2₁ crystal form (yellow cylinders) is aligned on one monomer of the tetramer structure to visualize the change in tilt angle of H12. In the tetrameric arrangement, H12 enables tighter packing between the monomers. BN-PAGE, Blue Native PAGE; DDM, n-dodecyl-β-D-maltoside; EM, electron microscopy; POT, proton-dependent oligopeptide transporter; SDS–PAGE, SDS–polyacrylamide gel electrophoresis.